Dengue disease poses a significant threat to over 2.5 billion people worldwide and currently, no licensed vaccine is available. An effective and safe vaccine requires a balanced immune response to all four serotypes of dengue viruses (DENV-1 to -4) because sequential infection by different dengue virus serotypes can lead to life-threatening dengue hemorrhagic fever or dengue shock syndrome. Live attenuated or chimeric virus-based vaccines are promising but they struggle with inadequate attenuation or an imbalanced immune response due to interference. While interference can be overcome by a 12-month immunization schedule, this schedule renders the vaccine unsuitable for recipients requiring rapid development of immunity. Additionally, safety concerns associated with 17D may limit its use in immunocompromised individuals, children or elderly. We have developed a series of flavivirus vaccines based on our clinically proven vaccine platform in three formats, which differ in the site of antigen attachment to the flagellin (a TLR5 ligand). These vaccine candidates can be efficiently produced and elicit protective levels of neutralizing antibodies to both dengue serotypes. TLR signaling triggers an innate immune cascade that enhances antigen uptake and presentation, and facilitates induction of adaptive immune response, thereby eliminating the need for adjuvants typically used in subunit vaccines. Flagellin contains of four distinct domains (D0, D1, D2, and D3) where D1 contains the TLR5 binding site. Antigens can be fused to the C-terminus in the C-term format, in place of D3 in the R3 format, or to both positions in two copies in the R3.2x formats. Among the three, the R3.2x format was found to be the safest and the most immunogenic in Phase I trials of influenza vaccine. To develop flavivirus vaccines, we initially fused the domain III of the envelope (EIII) to the C-terminus of the flagellin. We have demonstrated that monovalent West Nile and DENV-2 vaccine candidates based on the flagellin-EIII fusion induce potent antibody responses and are efficacious in mouse challenge models. We have subsequently generated flagellin-EIII dengue vaccines in various formats, and have identified R3.2x as the lead format for use in a tetravalent vaccine candidate in mice and non-human primates (NHP). Based on recent findings of neutralizing epitopes of human polyclonal and monoclonal antibodies, we have generated backup subunit flagellin-E vaccines that include major neutralizing epitopes in the domains III and I of the E protein (EIEIII) as well as the EI/EII junction region. We here propose to optimize the lead TDV formulation (dose level and component ratio) in immunogenicity studies in mice, and then to optimize the fermentation conditions and purification process for those candidates. We will next evaluate the immunogenicity and efficacy of the lead TDV in NHP DENV challenge models, followed by evaluation of dose range using a well-established rabbit reactogenicity model as well as formulation development. We will demonstrate that the tetravalent vaccine induces durable, balanced immune responses that are capable of neutralizing a broad spectrum of strains and genotypes of each serotype, and provides protection against all four DENV serotypes in mouse and monkey models. Once the lead candidates are locked we will develop a liquid formulation suitable for 4 C storage for at least one year. Further, we will confirm a safe and effective window of the TDV candidate to guide the dose selection in the subsequent GLP rabbit toxicity study as well as dose escalating study in phase I trial. The positive outcome of this study will be a solid step towards the successful development of a safer, cheaper, and effective vaccine, which can be used to prevent DENV-associated diseases. Lastly, VaxInnate's rapid and low cost bacteria-based manufacturing can easily be transferred to endemic countries as demonstrated by a recent successful process transfer of the 2009 H1N1 pandemic vaccine.